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Synergistic factors can enhance the toxicity of Bt toxins and delay the development of Bt resistance. Previous research has demonstrated that a Helicoverpa armigera cadherin fragment (HaCad-TBR) increased the toxicity of Cry1Ac in Plutella xylostella larvae but did not have a synergistic effect on Cry1B, Cry1C, and Cry1F toxins. In this study, a fusion protein (HaCad-TBR-2D3 V L ) derived from HaCad-TBR and a Bt Cry1-specific antibody peptide was expressed in Escherichia coli . The HaCad-TBR-2D3 V L enhanced Cry1Ac toxicity more efficiently in insects and Sf9 cells than HaCad-TBR and also significantly increased the toxicity of Cry1B, Cry1C, and Cry1F toxins in insects. Further investigation indicated that the improved stability in insect midguts and higher binding capacity with Bt toxins contributed to the enhanced synergism of HaCad-TBR-2D3 V L over HaCad-TBR. This study suggested that Bt antibody fragments can potentially broaden the synergistic range of Bt receptor fragments, providing a theoretical foundation for developing broad-spectrum synergists for other biopesticides.

Introduction: Human immunodeficiency virus type 1 (HIV) transmission mostly occurs through the genital and intestinal mucosae. Although HIV-1 transmission has been extensively investigated, gaps remain in understanding the initial steps of HIV entry through the colonic mucosa. We previously showed that HIV can selectively trigger mononuclear phagocytes (MNP) to migrate within colonic epithelial cells to sample virions. Mucosal exposure to human seminal plasma (HSP), rich in pro- and anti-inflammatory cytokines, chemokines and growth factors, may as well induce alterations of the colonic mucosa and recruit immune cells, hence, affecting pathogen sampling and transmission., Methods: Here, we studied the role of HSP on the paracellular intestinal permeability by analyzing the distribution of two proteins known to play a key role in controlling the intestinal barrier integrity, namely the tight junctions-associated junctional adhesion molecule (JAM-A) and the adherents junction associated protein E-cadherin (E-CAD), by immunofluorescence and confocal microscopy. Also, we evaluated if HSP promotes the recruitment of MNP cells, specifically, the CD11c and CD64 positive MNPs, to the apical side of the human colonic mucosa. At this scope, HSP of HIV-infected and uninfected individuals with known fertility status was tested for cytokines, chemokines and growth factors concentration and used in an ex vivo polarized colonic tissue culture system to mimic as closely as possible the physiological process., Results: HSP showed statistically significant differences in cytokines and chemokines concentrations between the three groups of donors, i.e. HIV infected, or uninfected fertile or randomly identified. Nevertheless, we showed that in the ex vivo tissue culture HSP in general, neither affected the morphological structure of the colonic mucosa nor modulated the paracellular intestinal permeability. Interestingly, CD11c+ MNP cells migrated to the apical surface of the colonic epithelium regardless, if incubated with HIV-infected or -uninfected HSPs, while CD64+ MNP cells, did not change their distribution within the colonic mucosa., Discussion: In conclusion, even if HSP did not perturb the integrity of the human colonic mucosa, it affected the migration of a specific subset of MNPs that express CD11c towards the apical side of the colonic mucosa, which in turn may be involved in pathogen sampling., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Baratella, Iannone, Cavarelli, Foglieni, Viganò, Moog, Elmore, Nozza, Alfano, Salonia, Dispinseri and Scarlatti.)

E-cadherin (Ecad) is an essential cell-cell adhesion protein with tumor suppression properties. The adhesive state of Ecad can be modified by the monoclonal antibody 19A11, which has potential applications in reducing cancer metastasis. Using X-ray crystallography, we determine the structure of 19A11 Fab bound to Ecad and show that the antibody binds to the first extracellular domain of Ecad near its primary adhesive motif: the strand-swap dimer interface. Molecular dynamics simulations and single-molecule atomic force microscopy demonstrate that 19A11 interacts with Ecad in two distinct modes: one that strengthens the strand-swap dimer and one that does not alter adhesion. We show that adhesion is strengthened by the formation of a salt bridge between 19A11 and Ecad, which in turn stabilizes the swapped β-strand and its complementary binding pocket. Our results identify mechanistic principles for engineering antibodies to enhance Ecad adhesion.

The mechanically activated Piezo channel plays a versatile role in conferring mechanosensitivity to various cell types. However, how it incorporates its intrinsic mechanosensitivity and cellular components to effectively sense long-range mechanical perturbation across a cell remains elusive. Here we show that Piezo channels are biochemically and functionally tethered to the actin cytoskeleton via the cadherin-β-catenin mechanotransduction complex, whose perturbation significantly impairs Piezo-mediated responses. Mechanistically, the adhesive extracellular domain of E-cadherin interacts with the cap domain of Piezo1, which controls the transmembrane gate, while its cytosolic tail might interact with the cytosolic domains of Piezo1, which are in close proximity to its intracellular gates, allowing a direct focus of adhesion-cytoskeleton-transmitted force for gating. Specific disruption of the intermolecular interactions prevents cytoskeleton-dependent gating of Piezo1. Thus, we propose a force-from-filament model to complement the previously suggested force-from-lipids model for mechanogating of Piezo channels, enabling them to serve as versatile and tunable mechanotransducers., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Immune checkpoint inhibitors prolong the survival of non-small cell lung cancer (NSCLC) patients. Although it has been acknowledged that there is some correlation between the efficacy of anti-programmed cell death-1 (PD-1) antibody therapy and immunohistochemical analysis, this technique is not yet considered foolproof for predicting a favorable outcome of PD-1 antibody therapy. We aimed to predict the efficacy of nivolumab based on a comprehensive analysis of RNA expression at the gene level in advanced NSCLC., Methods: This was a retrospective study on patients with NSCLC who were administered nivolumab at the Kansai Medical University Hospital. To identify genes associated with response to anti-PD-1 antibodies, we grouped patients into responders (complete and partial response) and non-responders (stable and progressive disease) to nivolumab therapy. Significant genes were then identified for these groups using Welch's t-test., Results: Among 42 analyzed cases (20 adenocarcinomas and 22 squamous cell carcinomas), enhanced expression of MAGE-A4, BBC3, and OTOA genes was observed in responders with adenocarcinoma, and enhanced expression of DAB2, HLA-DPB,1 and CDH2 genes was observed in responders with squamous cell carcinoma., Conclusions: This study predicted the efficacy of nivolumab based on a comprehensive analysis of mRNA expression at the gene level in advanced NSCLC. We also revealed different gene expression patterns as predictors of the effectiveness of anti PD-1 antibody therapy in adenocarcinoma and squamous cell carcinoma., (© 2022. The Author(s).)

Crosslinking of FcεRI-bound IgE triggers the release of a large number of biologically active, potentially anaphylactic compounds by mast cells. FcεRI activation ought to be well-controlled to restrict adverse activation. As mast cells are embedded in tissues, adhesion molecules may contribute to limiting premature activation. Here, we report that E-Cadherin serves that purpose. Having confirmed that cultured mast cells express E-Cadherin, a mast-cell-specific E-Cadherin deficiency, Mcpt5-Cre E-Cdh fl/fl mice, was used to analyze mast cell degranulation in vitro and in vivo. Cultured peritoneal mast cells from Mcpt5-Cre E-Cdh fl/fl mice were normal with respect to many parameters but showed much-enhanced degranulation in three independent assays. Soluble E-Cadherin reduced the degranulation of control cells. The release of some newly synthesized inflammatory cytokines was decreased by E-Cadherin deficiency. Compared to controls, Mcpt5-Cre E-Cdh fl/fl mice reacted much stronger to IgE-dependent stimuli, developing anaphylactic shock. We suggest E-Cadherin-mediated tissue interactions restrict mast cell degranulation to prevent their precocious activation., (© 2021 The Authors. European Journal of Immunology published by Wiley-VCH GmbH.)

Antibodies provide the functional biospecificity that has enabled the development of sensors, diagnostic tools, and assays in both laboratory and clinical settings. However, as multimarker screening becomes increasingly necessary due to the heterogeneity and complexity of human pathology, new methods must be developed that are capable of coordinating the precise assembly of multiple, distinct antibodies. To address this technological challenge, we engineered a bottom-up, high-throughput method in which DNA patterns, comprising unique 20-base pair oligonucleotides, are patterned onto a substrate using photolithography. These microfabricated surface patterns are programmed to hybridize with, and instruct the multiplexed assembly of, antibodies conjugated with the complementary DNA strands. We demonstrate that this simple, yet robust, approach preserves the antibody-binding functionality in two common applications: antibody-based cell capture and label-free surface marker screening. Using a simple proof-of-concept capture device, we achieved high purity separation of a breast cancer cell line, MCF-7, from a blood cell line, Jurkat, with capture purities of 77.4% and 96.6% when using antibodies specific for the respective cell types. We also show that antigen-antibody interactions slow cell trajectories in flow in the next-generation microfluidic node-pore sensing (NPS) device, enabling the differentiation of MCF-7 and Jurkat cells based on EpCAM surface-marker expression. Finally, we use a next-generation NPS device patterned with antibodies against E-cadherin, N-cadherin, and β-integrin-three markers that are associated with epithelial-mesenchymal transitions-to perform label-free surface marker screening of MCF10A, MCF-7, and Hs 578T breast epithelial cells. Our high-throughput, highly versatile technique enables rapid development of customized, antibody-based assays across a host of diverse diseases and research thrusts.

ADAM15 is highly expressed in malignant tumors and is correlated with tumor progression. However, the role of ADAM15 in hepatocellular carcinoma (HCC) remains unclear. In the study, our results indicated that ADAM15 was highly expressed in HCC tissues and cells compared with corresponding tissues and liver cells. Overexpression of ADAM15 was linked to poor prognosis, and was an independent risk factor for HCC prognosis. Besides, analysis of immune infiltration indicated that ADAM15 expression was related to tumor infiltrating lymphocytes based on the TIMER, TISIDB and GEPIA databases. Many immune checkpoint gene expression was associated with ADAM15 expression. Functional enrichment analyses indicated that apoptosis, cell adhesion was enriched. ADAM15 knockdown promoted apoptosis and suppressed proliferation, migration and invasion of liver cancer cells. The findings of western blot showed that ADAM15 knockdown reduced the expression of Bcl-2, Vimentin, N-Cadherin and Snail, and elevated the expression of Bax, E-cadherin and ZO-1. However, overexpression of ADAM15 had the opposite results. Collectively, our findings demonstrated that ADAM15 was connected with poor prognosis of HCC patients, and could be considered as a potential biomarker for the diagnosis and treatment of HCC.

Epithelial-mesenchymal transition (EMT) is an essential biological process involved in the initiation and progression of cancer by which epithelial tumor cells lose their differentiated characteristics, such as cell-cell adhesion and apical-basal polarity and acquire a more invasive and∕or metastatic mesenchymal phenotype. The present study investigated the expression of immunomarkers with a role in EMT of non-melanoma skin cancers (NMSCs), such as E-cadherin, fibronectin and Slug, for a number of 50 NMSCs, represented by 30 cases of basal cell carcinomas (BCCs) and 20 cases of squamous cell carcinomas (SCCs). For BCC, the statistical analysis of the investigated immunomarkers indicated significantly differences in relation to the depth of invasion, and for E-cadherin and fibronectin with the degree of risk. In the case of SCC, the statistical analysis indicated significant differences of E-cadherin and Slug with the degree of tumor differentiation, and for fibronectin and Slug with the depth of invasion. The analysis of the distribution for the percentage values of the investigated immunomarkers in the case of BCC indicated a significant negative linear relation between E-cadherin/fibronectin and E-cadherin/Slug, and in SCC a significant negative linear relation between E-cadherin∕fibronectin, E-cadherin∕Slug and a positive linear one in the case of fibronectin∕Slug. The study indicates through the statistically significant relation between E-cadherin∕fibronectin and E-cadherin∕Slug, the EMT intervention in carcinogenesis of NMSC.

Cell-cell junctions are critical for the maintenance of cellular as well as tissue polarity and integrity. Dysfunction of airway epithelial barrier has been shown to be involved in the pathogenesis of acute lung injury (ALI). Yet the role of phosphatidylinositol 3-kinase delta (PI3Kδ) in dysregulation of airway epithelial barrier integrity in ALI has not been addressed. Mice were subjected to intratracheal instillation of lipopolysaccharide (LPS) to generate a ALI model. Two pharmacological inhibitors of PI3Kδ, IC87114 and AMG319, were respectively given to the mice. Expression of p110δ and its downstream substrate phospho-AKT (Ser473) was increased in LPS-exposed lungs. These increases were inhibited by IC87114 or AMG319. LPS led to pronounced lung injury that was accompanied by significant airway neutrophil recruitment and bronchial epithelial morphological alterations 72 h after exposure. We also found compromised expression of adherens junction protein E-cadherin and tight junction protein claudin-2 in the airway epithelial cells. Treatment with either IC87114 or AMG319 not only attenuated LPS-induced edema, lung injury and neutrophilc inflammation, reduced total protein concentration and IL-6, TNF-α secretion in BALF, but also restored epithelial E-cadherin and claudin-2 expression. In summary, our results showed that LPS can induce a delayed effect on airway epithelial barrier integrity that is mediated by PI3Kδ in a mouse model of ALI., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Cancer stemness and immune evasion are closely associated, and play critical roles in tumor development and resistance to immunotherapy. However, little is known about the underlying molecular mechanisms that coordinate this association. Here, it is reported that elevated circular RNA FAT1 (circFAT1) in squamous cell carcinoma (SCC) unifies and regulates the positive association between cancer stemness and immune evasion by promoting STAT3 activation. circFAT1 knockdown (KD) reduces tumorsphere formation of SCC cells in vitro and tumor growth in vivo. Bioinformatic analysis reveals that circFAT1 KD impairs the cancer stemness signature and activates tumor cell-intrinsic immunity. Mechanistically, circFAT1 binding to STAT3 in the cytoplasm prevents STAT3 dephosphorylation by SHP1 and promotes STAT3 activation, resulting in inhibition of STAT1-mediated transcription. Moreover, circFAT1 KD significantly enhances PD1 blockade immunotherapy by promoting CD8 + cell infiltration into tumor microenvironment. Taken together, the results demonstrate that circFAT1 is an important regulator of cancer stemness and antitumor immunity., Competing Interests: The authors declare no conflict of interest., (© 2021 The Authors. Advanced Science published by Wiley‐VCH GmbH.)

Membranous nephropathy (MN) occurs due to deposition of immune complexes along the subepithelial region of glomerular basement membrane. Two previously identified target antigens for the immune complexes, PLA2R (identified in 2009) and THSD7A (in 2014), account for approximately 60% of all MN, both primary and secondary. In the remaining MN, target antigens were unknown. Use of laser microdissection and mass spectrometry enabled identification of new "antigens." This approach led to the identification of four novel types of MN: exotosin 1 (EXT1)- and exotosin 2 (EXT2)-associated MN, NELL1-associated MN, Sema3B-associated MN, and PCDH7-associated MN. Each of these represents a distinct disease entity, with different clinical and pathologic findings. In this review, the structure of the proteins and the clinical and pathologic findings of the new types of MN are discussed. The role of mass spectrometry for accurate diagnosis of MN cannot be overemphasized. Finally, any classification of MN should be made on the basis of the antigens that are detected. Further studies are required to understand the pathophysiology, response to treatment, and outcomes of these new MNs., (Copyright © 2021 by the American Society of Nephrology.)

P-cadherin is overexpressed in various cancers and can be a target for radioimmunotherapy. We investigated the preclinical pharmacokinetics and pharmacology of FF-21101, an 111 In- or 90 Y-conjugated monoclonal antibody against P-cadherin, to evaluate its clinical applications. Methods: The radiochemical purity, binding affinity, and in vitro serum stability of 111 In or 90 Y-labeled FF-21101 were evaluated. The pharmacokinetics of 111 In or 90 Y-FF-21101 were compared in normal mice. Tumor accumulation after 111 In-FF-21101 administration was investigated in mice bearing subcutaneous tumors with high (NCI-H1373), moderate (EBC-1), or no (A549) P-cadherin expression. The tumor suppression effect after a single intravenous injection of 90 Y-FF-21101 was assessed in NCI-H1373 and EBC-1 mouse xenograft models. The relationship between antibody dose and tumor accumulation was investigated in the NCI-H1373 mouse xenograft model. The absorbed radiation dose in humans after injection of 90 Y-FF-21101 was estimated using γ-camera images of cynomolgus monkeys. Results: The radiochemical purities of 111 In- and 90 Y-FF-21101 were 98.2% ± 2.5% ( n = 9) and 99.3% ± 0.6% ( n = 5), respectively. The dissociation constants were 1.083 nM for 111 In-FF-21101 and 1.367 nM for 90 Y-FF-21101. Both 111 In- and 90 Y-FF-21101 were stable in human serum after 96 h of incubation and exhibited similar pharmacokinetics in normal mice. The tumor accumulation of 111 In-FF-21101 was closely related to the intensity of P-cadherin expression in the cells. 90 Y-FF-21101 showed significant tumor growth inhibition, indicating that NCI-H1373 and EBC-1 recurrence was not observed after intravenous administration of 3.7 and 7.4 MBq, respectively of 90 Y-FF-21101 per animal. Tumor uptake in the mouse xenograft model and estimated absorbed radiation doses in the spleen of monkeys decreased with increasing antibody doses of 111 In-FF-21101. Conversely, the estimated absorbed radiation dose in the red marrow increased with increasing antibody dose. An antibody dose of 4.8 mg/m 2 was considered appropriate for humans, on the basis of efficacy and safety. The maximum tolerated administered activity of 90 Y-FF-21101 was estimated to be 2,886 MBq/human. Conclusion: FF-21101 radioimmunotherapy exhibited high antitumor affinity and antitumor efficacy in mouse xenograft models. Extrapolation of the pharmacokinetics in monkeys to humans suggests the potential for clinical application of FF-21101 for treating P-cadherin-expressing tumor., (© 2021 by the Society of Nuclear Medicine and Molecular Imaging.)

Epitope spreading is an important mechanism for the development of autoantibodies (autoAbs) in autoimmune diseases. The study of epitope spreading in human autoimmune diseases is limited due to the major challenge of identifying the initial/primary target epitopes on autoantigens in autoimmune diseases. We have been studying the development of autoAbs in an endemic human autoimmune disease, Brazilian pemphigus foliaceus (or Fogo Selvagem (FS)). Our previous findings demonstrated that patients before (i.e. preclinical) and at the onset of FS have antibody (Ab) responses against other keratinocyte adhesion molecules in addition to the main target autoantigen of FS, desmoglein 1 (Dsg1), and anti-Dsg1 monoclonal Abs (mAbs) cross-reacted with an environmental antigen LJM11, a sand fly saliva protein. Since sand fly is prevalent in FS endemic regions, individuals in these regions could develop Abs against LJM11. The anti-LJM11 Abs could recognize different epitopes on LJM11, including an epitope that shares the structure similarity with an epitope on Dsg1 autoantigen. Thus, Ab response against this epitope on LJM11 could be the initial autoAb response detected in individuals in FS endemic regions, including those who eventually developed FS. Accordingly, this LJM11 and Dsg1 cross-reactive epitope on Dsg1 could be the primary target of the autoimmune response in FS. This investigation aimed to determine whether the autoAb responses against keratinocyte adhesion molecules are linked and originate from the immune response to LJM11. The anti-Dsg1 mAbs from preclinical FS and FS individuals were employed to determine their specificity or cross-reactivity to LJM11 and keratinocyte adhesion molecules. The cross-reactive epitopes on autoantigens were mapped. Our results indicate that all tested mAbs cross-reacted with LJM11 and keratinocyte adhesion molecules, and we identified an epitope on these keratinocyte adhesion molecules which is mimicked by LJM11. Thus, the cross-reactivity could be the mechanism by which the immune response against an environmental antigen triggers the initial autoAb responses. Epitope spreading leads to the pathogenic autoAb development and ensuing FS among genetically susceptible individuals., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Introduction: An immunohistochemical study has occasionally been performed to diagnose anaplastic thyroid carcinoma (ATC). However, antibodies to confirm the undifferentiated nature of ATC have not yet been evaluated. The aim of this study was to evaluate E-cadherin and β-catenin expressions in immunoreactivity to determine undifferentiated carcinoma cells in the diagnosis of ATC., Methods: We immunohistochemically examined 29 ATCs, 30 poorly differentiated thyroid carcinomas (PDTCs), 22 well-differentiated thyroid carcinomas (WDTCs), and 3 squamous cell carcinomas. Antibodies for thyroid transcription factor-1 (TTF-1), paired-box gene 8 (PAX8), β-catenin, and E-cadherin were used., Results: All WDTCs tested positive for TTF-1, PAX8, and E-cadherin. The positive rates of TTF-1, PAX8, and E-cadherin were 93.3, 93.3, and 100%, respectively, in PDTCs and 17.2, 51.7, and 10.3%, respectively, in ATCs. WDTC expressed the lateral cell membrane staining for β-catenin and E-cadherin, whereas PDTC showed circumferential cell membranous expression (fishnet pattern). β-catenin cell membrane expression in ATCs is lost or discontinuous. Carcinoma cells with β-catenin nuclear expression without cell membranous expression were scattered in 72.4% of ATCs but were not observed in the other carcinomas., Conclusion: We propose 3 immunohistochemical findings to determine undifferentiated carcinoma cells in the diagnosis of ATC: (1) β-catenin nuclear expression with no or reduced cell membranous expression, (2) the loss or discontinuous pattern of E-cadherin expression, and (3) the loss of PAX8 nuclear expression., (© 2021 S. Karger AG, Basel.)

Free extracellular heme has been shown to activate several compartments of innate immunity, acting as a danger-associated molecular pattern (DAMP) in hemolytic diseases. Although localized endothelial barrier (EB) disruption is an important part of inflammation that allows circulating leukocytes to reach inflamed tissues, non-localized/deregulated disruption of the EB can lead to widespread microvascular hyperpermeability and secondary tissue damage. In mouse models of sickle cell disease (SCD), EB disruption has been associated with the development of a form of acute lung injury that closely resembles acute chest syndrome (ACS), and that can be elicited by acute heme infusion. Here we explored the effect of heme on EB integrity using human endothelial cell monolayers, in experimental conditions that include elements that more closely resemble in vivo conditions. EB integrity was assessed by electric cell-substrate impedance sensing in the presence of varying concentrations of heme and sera from SCD patients or healthy volunteers. Heme caused a dose-dependent decrease of the electrical resistance of cell monolayers, consistent with EB disruption, which was confirmed by staining of junction protein VE-cadherin. In addition, sera from SCD patients, but not from healthy volunteers, were also capable to induce EB disruption. Interestingly, these effects were not associated with total heme levels in serum. However, when heme was added to sera from SCD patients, but not from healthy volunteers, EB disruption could be elicited, and this effect was associated with hemopexin serum levels. Together our in vitro studies provide additional support to the concept of heme as a DAMP in hemolytic conditions., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Santaterra, Fiusa, Hounkpe, Chenou, Tonasse, da Costa, Garcia-Weber, Domingos, Lima, Borba-Junior, Araújo, Lucena-Araújo, Bezerra, dos Santos, Costa, Millán and De Paula.)

Purpose: 90 Y-FF-21101 is an Yttrium-90-conjugated, chimeric mAb that is highly specific for binding to human placental (P)-cadherin, a cell-to-cell adhesion molecule overexpressed and associated with cancer invasion and metastatic dissemination in many cancer types. We report the clinical activity of 90 Y-FF-21101 in a first-in-human phase I study in patients with advanced solid tumors., Patients and Methods: The safety and efficacy of 90 Y-FF-21101 were evaluated in a phase I 3+3 dose-escalation study in patients with advanced solid tumors ( n = 15) over a dose range of 5-25 mCi/m 2 . Dosimetry using 111 In-FF-21101 was performed 1 week prior to assess radiation doses to critical organs. Patients who demonstrated clinical benefit received repeated 90 Y-FF-21101 administration every 4 months., Results: 111 In-FF-21101 uptake was observed primarily in the spleen, kidneys, testes, lungs, and liver, with tumor uptake observed in the majority of patients. Organ dose estimates for all patients were below applicable limits. P-cadherin expression H-scores ranged from 0 to 242 with 40% of samples exhibiting scores ≥100. FF-21101 protein pharmacokinetics were linear with increasing antibody dose, and the mean half-life was 69.7 (±12.1) hours. Radioactivity clearance paralleled antibody clearance. A complete clinical response was observed in a patient with clear cell ovarian carcinoma, correlating with a high tumor P-cadherin expression. Stable disease was observed in a variety of other tumor types, without dose-limiting toxicity., Conclusions: The favorable safety profile and initial antitumor activity observed for 90 Y-FF-21101 warrant further evaluation of this radioimmunotherapeutic (RIT) approach and provide initial clinical data supporting P-cadherin as a potential target for cancer treatment., (©2020 American Association for Cancer Research.)

Cell-cell interaction in skin homeostasis is tightly controlled by adherens junctions (AJs). Alterations in such regulation lead to melanoma development. However, mutations in AJs and their functional consequences are still largely unknown. Methods: Cadherin mutations in skin cutaneous melanoma were identified using sequencing data from TCGA dataset, followed by cross-validation with data from non-TCGA cohorts. Mutations with significant occurrence were subjected to structural prediction using MODELLER and functional protein simulation using GROMACS software. Neo-antigen prediction was carried out using NetMHCpan tool. Cell-based fluorescence reporter assay was used to validate β-catenin activity in the presence of cadherin mutations. Clinical significance was analyzed using datasets from TCGA and other non-TCGA cohorts. Targeted gene exon sequencing and immunofluorescence staining on melanoma tissues were performed to confirm the in silico findings. Results: Highly frequent mutations in type-II classical cadherins were found in melanoma with one unique recurrent mutation (S524L) in the fifth domain of CDH6, which potentially destabilizes Ca 2+ -binding and cell-cell contacts. Mutational co-occurrence and physical dynamics analyses placed CDH6 at the center of the top-four mutated cadherins (core CDHs; all type-II), suggesting altered heterophilic interactions in melanoma development. Mutations in the intracellular domains significantly disturbed CDH6/β-catenin complex formation, resulting in β-catenin translocation into cytosol or nucleus and dysregulation of canonical Wnt/β-catenin signaling. Although mutations in core CDH genes correlated with advanced cancer stages and lymph node invasion, the overall and disease-free survival times in those patients were longer in patients with wild-type. Peptide/MHC-I binding affinity predictions confirmed overall increased neo-antigen potentials of mutated cadherins, which associated with T-lymphocyte infiltration and better clinical outcomes after immunotherapy. Conclusion: Changes in cell-cell communications by somatic mutations in AJ cadherins function as one of mechanisms to trigger melanoma development. Certain mutations in AJs may serve as potential neo-antigens which conversely benefit patients for longer survival times., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Quantitatively determining in vivo achievable drug concentrations in targeted organs of animal models and subsequent target engagement confirmation is a challenge to drug discovery and translation due to lack of bioassay technologies that can discriminate drug binding with different mechanisms. We have developed a multiplexed and high-throughput method to quantify drug distribution in tissues by integrating high content screening (HCS) with U-Net based deep learning (DL) image analysis models. This technology combination allowed direct visualization and quantification of biologics drug binding in targeted tissues with cellular resolution, thus enabling biologists to objectively determine drug binding kinetics.

Objectives: Alloyed metallic nanoparticles of silver and copper are effective against intracellular infection. However, systemic toxicity may arise due to the non-specific delivery of the nanoparticles. In addressing the issue, this study deals with the targeting of silver-copper-boron (ACB) nanoparticles to infected osteoblasts, which could decrease systemic toxicity and form the basis of targeting specific markers expressed in bone infections., Methods: ACB nanoparticles were synthesized and conjugated to the Cadherin-11 antibody (OBAb). The effect of targeting nanoparticles against extracellular and intracellular S. aureus was determined by enumeration of bacterial growth. The binding of the targeting nanoparticles to infected osteoblasts as well as the visualization of live/dead bacteria due to treatment was carried out using fluorescence microscopy. MTT assay was used to determine the viability of osteoblasts with different concentrations of the nanoparticles., Results: The ACB nanoparticles conjugated to OBAb (ACB-OBAb) were effective against extracellular S. aureus. The ACB-OBAb nanoparticles showed a 1.32 log reduction of intracellular S. aureus at a concentration of 1mg/L. The ACB-OBAb nanoparticles were able to bind to the infected osteoblast and showed toxicity to osteoblasts at levels ≥20mg/L. Also, the percentage of silver, copper, and boron in the nanoparticles determined the effectiveness of their antibacterial activity., Conclusion: The ACB-OBAb nanoparticles were able to target the osteoblasts and demonstrated significant antibacterial activity against intracellular S. aureus. Targeting shows promise as a strategy to target specific markers expressed on infected osteoblasts for efficient nanoparticle delivery, and further animal studies are recommended to test its efficacy in vivo., Competing Interests: The authors have declared that no competing interests exist.

Type I interferons (IFNs) are the first line of defense against viral infection. Using a mouse model of influenza A virus infection, we found that IFN-κ was one of the earliest responding type I IFNs after infection with H9N2, a low-pathogenic avian influenza A virus, whereas this early induction did not occur upon infection with the epidemic-causing H7N9 virus. IFN-κ efficiently suppressed the replication of various influenza viruses in cultured human lung cells, and chromodomain helicase DNA binding protein 6 (CHD6) was the major effector for the antiviral activity of IFN-κ, but not for that of IFN-α or IFN-β. The induction of CHD6 required both of the type I IFN receptor subunits IFNAR1 and IFNAR2, the mitogen-activated protein kinase (MAPK) p38, and the transcription factor c-Fos but was independent of signal transducer and activator of transcription 1 (STAT1) activity. In addition, we showed that pretreatment with IFN-κ protected mice from lethal influenza viral challenge. Together, our findings identify an IFN-κ-specific pathway that constrains influenza A virus and provide evidence that IFN-κ may have potential as a preventative and therapeutic agent against influenza A virus., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Altered intestinal epithelial integrity is an important susceptibility trait in inflammatory bowel disease (IBD), and early life stressors are reported to contribute to this disease susceptibility in adulthood. To identify disease mechanisms associated with early-life trauma that exacerbate IBD in adulthood, we used a "double-hit" neonatal inflammation (NI) and adult inflammation (AI) model that exhibits more severe mucosal injury in the colon later in life. In this study, we explore the underlying mechanisms of this aggravated injury. In rats exposed to both NI and AI, we found sustained increases in colonic permeability accompanied by significantly attenuated expression of the epithelial junction protein E-cadherin. Quantitative RT-PCR revealed a decreased Cdh1 (gene of E-cadherin) mRNA expression in NI + AI rats compared with NI or AI rats. Next, we performed microRNA microarrays to identify potential regulators of E-cadherin in NI + AI rats. We confirmed the overexpression of miR-155, a predicted regulator of E-cadherin, and selected it for further analysis based on reported significance in human IBD. Using ingenuity pathway analysis software, the targets and related canonical pathway of miR-155 were analyzed. Mechanistic studies identified histone hyperacetylation at the Mir155 promoter in NI + AI rats, concomitant with elevated RNA polymerase II binding. In vitro, E-cadherin knockdown markedly increased epithelial cell permeability, as did overexpression of miR-155 mimics, which significantly suppressed E-cadherin protein. In vivo, NI + AI colonic permeability was significantly reversed with administration of miR-155 inhibitor rectally. Our collective findings indicate that early-life inflammatory stressors trigger a significant and sustained epithelial injury by suppressing E-cadherin through epigenetic mechanisms., (Copyright © 2020 by The American Association of Immunologists, Inc.)

One of the most promising strategies to treat cancer is the use of therapeutic antibodies that disrupt cell-cell adhesion mediated by dysregulated cadherins. The principal site where cell-cell adhesion occurs encompasses Trp2 found at the N-terminal region of the protein. Herein, we employed the naturally exposed highly conserved peptide Asp1-Trp2-Val3-Ile4-Pro5-Pro6-Ile7, as epitope to prepare molecularly imprinted polymer nanoparticles (MIP-NPs) to recognize cadherins. Since MIP-NPs target the site responsible for adhesion, they were more potent than commercially available therapeutic antibodies for inhibiting cell-cell adhesion in cell aggregation assays, and for completely disrupting three-dimensional tumor spheroids as well as inhibiting invasion of HeLa cells. These biocompatible supramolecular anti-adhesives may potentially be used as immunotherapeutic or sensitizing agents to enhance antitumor effects of chemotherapy., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Objective: Cadherin-17 (CDH17) is a transmembrane protein that mediates cell-cell adhesion and is frequently expressed in adenocarcinomas, including gastric cancer. CDH17 may be an effective diagnostic marker for the staging of gastric cancer. Here, we developed an 111 In-labeled anti-CDH17 monoclonal antibody (Mab) as an imaging tracer and performed biodistribution and single-photon emission computed tomography (SPECT)/computed tomography (CT) imaging studies using mice with CDH17-positive gastric cancer xenografts. CDH17 expression in gastric cancer specimens was also analyzed., Methods: The cross-reactivity and affinity of our anti-CDH17 Mab D2101 was evaluated by surface plasmon resonance analysis and cell enzyme-linked immunosorbent assay, respectively. Biodistribution and SPECT/CT studies of 111 In-labeled D2101 ( 111 In-D2101) were performed. CDH17 expression in gastric cancer specimens was evaluated by immunohistochemistry., Results: Surface plasmon resonance analysis revealed that D2101 specifically recognizes human CDH17, but not murine CDH17. The affinity of D2101 slightly decreased as a result of the radiolabeling procedures. The biodistribution study revealed high uptake of 111 In-D2101 in tumors (maximum, 39.2 ± 9.5% ID/g at 96 h postinjection), but low uptake in normal organs, including the stomach. Temporal SPECT/CT imaging with 111 In-D2101 visualized tumors with a high degree of tumor-to-nontumor contrast. Immunohistochemical analysis revealed that, compared with HER2, which is a potential marker of N-stage, CDH17 had a higher frequency of positivity in specimens of primary and metastatic gastric cancer., Conclusion: Our 111 In-anti-CDH17 Mab D2101 depicted CDH17-positive gastric cancer xenografts in vivo and has the potential to be an imaging probe for the diagnosis of primary lesions and lymph-node metastasis in gastric cancer.

Recent studies have indicated that Macrophage migration inhibitory factor (MIF) plays an important role in the prevention and treatment of asthma. However the role of MIF in airway inflammation and airway epithelial barrier disruption in house dust mite (HDM)-induced asthma has not been addressed. We hypothesized that MIF contributed to HDM-induced the production of Th2-associated cytokines and E-cadherin dysfunction in asthmatic mice and 16HBE cells. In vivo, a HDM-induced asthma mouse model was set up and mice treated with MIF antagonist ISO-1 after HDM. The mice treated with the ISO-1 ameliorated airway hyper-reactivity, airway inflammation, increased serum IgE levels, the aberrant arrangement of E-cadherin as well as the release of Th2 cytokines induced by HDM. In vitro, the exposure of 16HBE cells to HDM and rhMIF resulted in airway epithelial barrier disruption, inflammatory cytokine production and enhanced glycolytic flux. While these changes were attenuated by MIF siRNA treatment. Sequentially, treatment of 16HBE cells with PFKFB3 antagonist PFK15 significantly lowered rhMIF-induced these changes in 16HBE cells. Therefore, these results indicate that MIF may be an important contributor in airway inflammation and airway epithelial barrier disruption of HDM-induced asthma. Moreover, HDM specifically induces airway inflammation and airway epithelial barrier disruption of 16HBE cells through MIF-mediated enhancement of aerobic glycolysis., (Copyright © 2019. Published by Elsevier Inc.)

Leptospirosis is a worldwide zoonosis caused by pathogenic species of Leptospira . Leptospires are able to adhere to exposed extracellular matrix in injured tissues and, once in the bloodstream, can survive the attack of the immune system and spread to colonize target organs. In this work, we report that two novel putative proteins, coded by the genes LIC11711 and LIC12587 of L. interrogans serovar Copenhageni are conserved among pathogenic strains, and probably exposed in the bacterial surface. Soluble recombinant proteins were expressed in Escherichia coli , purified and characterized. Both recombinant proteins bound to laminin and E-cadherin, suggesting an initial adhesion function in host epithelial cells. The recombinant protein LIC11711 (rLIC11711) was able to capture plasminogen (PLG) from normal human serum and convert to enzymatically active plasmin (PLA), in the presence of PLG activator. rLIC12587 (recombinant protein LIC12587) displayed a dose dependent and saturable interaction with components C7, C8, and C9 of the complement system, reducing the bactericidal effect of the complement. Binding to C9 may have consequences such as C9 polymerization inhibition, interfering with the membrane attack complex formation. Blocking LIC11711 and LIC12587 on bacterial cells by the respective antiserum reduced leptospiral cell viability when exposed to normal human serum (NHS). Both recombinant proteins could be recognized by serum samples of confirmed leptospirosis, but not of unrelated diseases, suggesting that the native proteins are immunogenic and expressed during leptospirosis. Taken together, our data suggest that these proteins may have a role in leptospiral pathogenesis, participating in immune evasion strategies.

Adiponectin exerts beneficial pleiotropic effects through three receptors, AdipoR1, AdipoR2, and T-cadherin; it also exerts immunomodulatory effects. We previously demonstrated that adiponectin levels are altered in common variable immunodeficiency disease (CVID). The purpose of the present study was to investigate further the specific involvement of adiponectin in CVID by characterizing (i) the expression profile of adiponectin receptors on peripheral blood mononuclear cells; (ii) the levels of another relevant adipokine, namely leptin; (iii) the levels of five other cytokines (IL-2, IL-6, IL-10, TNFα, and IFNγ) in 24 patients on maintenance therapy, in 18 treatment-naïve patients (before and 24 h after the first Ig infusion) and in 28 healthy controls. We found that (i) adiponectin was down-expressed in patients on maintenance therapy and in treatment-naïve patients, and that it increased in treatment-naïve patients 24 h after the first Ig infusion; (ii) leptin expression did not differ between maintenance patients and controls either before or after the first Ig infusion; (iii) AdipoR1 expression was significantly higher on B lymphocytes, monocytes and NK cells of CVID patients than in controls; (iv) the expression of AdipoR1 and AdipoR2 on B lymphocytes, monocytes and NK cells was higher after the first Ig infusion than in treatment-naïve patients; (v) T-cadherin expression did not differ between treatment- naïve CVID patients and controls, and was not affected by Ig infusion; and (vi) IL-6, IL-8, IL-10, and TNFα levels were differently expressed in CVID patients on therapy maintenance and were not affected by the first Ig replacement therapy. This is the first study to demonstrate that the expression of AdipoRs in peripheral blood mononuclear cells from CVID patients differs from that of controls, and changes after the first Ig infusion. The specificity of adiponectin involvement in CVID is supported by the absence of changes in leptin levels and in the levels of the cytokines investigated. Taken together, these results suggest that the adiponectin system plays an important and specific role in CVID. A better understanding of adiponectin as a link in the cross-talk between the immune system and adipose tissue may provide additional benefits for the management of CVID patients., (Copyright © 2019 Polito, Nigro, Pecoraro, Monaco, Perna, Sanduzzi, Genovese, Spadaro and Daniele.)

Inflammatory bowel disease (IBD) is a chronic inflammatory disorder with rising incidence. Diseased tissues are heavily vascularized. Surprisingly, the pathogenic impact of the vasculature in IBD and the underlying regulatory mechanisms remain largely unknown. IFN-γ is a major cytokine in IBD pathogenesis, but in the context of the disease, it is almost exclusively its immune-modulatory and epithelial cell-directed functions that have been considered. Recent studies by our group demonstrated that IFN-γ also exerts potent effects on blood vessels. Based on these considerations, we analyzed the vessel-directed pathogenic functions of IFN-γ and found that it drives IBD pathogenesis through vascular barrier disruption. Specifically, we show that inhibition of the IFN-γ response in vessels by endothelial-specific knockout of IFN-γ receptor 2 ameliorates experimentally induced colitis in mice. IFN-γ acts pathogenic by causing a breakdown of the vascular barrier through disruption of the adherens junction protein VE-cadherin. Notably, intestinal vascular barrier dysfunction was also confirmed in human IBD patients, supporting the clinical relevance of our findings. Treatment with imatinib restored VE-cadherin/adherens junctions, inhibited vascular permeability, and significantly reduced colonic inflammation in experimental colitis. Our findings inaugurate the pathogenic impact of IFN-γ-mediated intestinal vessel activation in IBD and open new avenues for vascular-directed treatment of this disease.

We present the guideline for use of yttrium-90-labeled anti-P-cadherin antibody injection for radionuclide therapy in clinical trials on the basis of radiation safety issues in Japan. This guideline was prepared by a study supported by the Ministry of Health, Labour, and Welfare, and approved by the Japanese Society of Nuclear Medicine. Treatment using yttrium-90-labeled anti-P-cadherin antibody injection in Japan should be carried out according to this guideline. Although this guideline is applied in Japan, the issues for radiation protection shown here are considered internationally useful as well. Only the original Japanese version is the formal document.

Primary Sjögren's syndrome (pSS) is a chronic inflammatory, autoimmune and systemic disorder commonly associated with dry eyes and a dry mouth. Recently, the hypothetical link between epithelial-mesenchymal transition (EMT)-dependent salivary gland (SG) fibrosis and chronic inflammatory conditions has been suggested. In this study, we present data demonstrating a negative correlation of the epithelial marker E-cadherin expression and a positive correlation of mesenchymal vimentin and collagen type I expression with increasing degrees of tissue inflammation in pSS SG specimens. In addition, as it is not clear whether dysregulated cytokines in pSS, interleukin (IL)-17 and IL-22 may also contribute to the EMT-dependent fibrosis process, the effect of IL-17 and IL-22 treatment on EMT-dependent SG fibrosis was evaluated in primary human salivary gland epithelial cells (SGEC) isolated from healthy subjects. Here we present data demonstrating that IL-17 and IL-22 can induce SGEC to undergo a morphological and phenotypical transition to a mesenchymal phenotype. In support of this, vimentin and collagen type I were up-regulated while a decreased expression of E-cadherin occurs after interleukin treatment, and co-operation between IL-17 and Il-22 was required to induce the EMT., (© 2019 British Society for Immunology.)

Precise integration of individual cell behaviors is indispensable for collective tissue morphogenesis and maintenance of tissue integrity. Organized multicellular behavior is achieved via mechanical coupling of individual cellular contractility, mediated by cell adhesion molecules at the cell-cell interface. Conventionally, gene depletion or laser microsurgery has been used for functional analysis of intercellular mechanotransduction. Nevertheless, these methods are insufficient to investigate either the spatiotemporal dynamics or the biomolecular contribution in cell-cell mechanical coupling within collective multicellular behaviors. Herein, we present our effort in adaption of PhoCl for attenuation of cell-to-cell tension transmission mediated by E-cadherin. To release intercellular contractile tension applied on E-cadherin molecules with external light, a genetically encoded photocleavable module called PhoCl was inserted into the intracellular domain of E-cadherin, thereby creating photocleavable cadherin (PC-cadherin). In response to light illumination, the PC-cadherin cleaved into two fragments inside cells, resulting in attenuating mechanotransduction at intercellular junctions in living epithelial cells. Light-induced perturbation of the intercellular tension balance with surrounding cells changed the cell shape in an epithelial cell sheet. The method is expected to enable optical manipulation of force-mediated cell-to-cell communications in various multicellular behaviors, which contributes to a deeper understanding of embryogenesis and oncogenesis.

Objective: Vascular endothelial (VE)-cadherin-mediated adherens junction is critical to maintain endothelial integrity. Besides its role of homophilic intercellular adhesion, VE-cadherin also has a role of outside-in signaling with functional consequences for vascular physiology. However, the nature of these signals remains not completely understood., Materials and Methods: Human umbilical vein endothelial cells (HUVECs) were used in cell culture experiments. Confluent HUVECs were treated with VE-cadherin function-blocking antibodies BV9 (50 μg/ml) or IgG control. Antibody array was used to screen for cytokine/chemokine in supernatant. For VE-cadherin knockdown, siRNA transfection was used. ELISA, Western blot, and qRT-PCR were used to confirm the expression of screened cytokine/chemokine. To explore the possible mechanisms, Scr phosphorylation was detected and Scr inhibitor PP2 (1 μM) was used. To investigate in vivo relevance of the findings, BV9 and the indicated neutralizing antibodies were injected into mice and then lung vascular leak and inflammation were examined by Evans blue assay and lung tissue H&E, respectively., Results: Using a non-biased, high-throughout human cytokine/chemokine antibody array, we first found that disruption of VE-cadherin-mediated adhesion by function-blocking antibody BV9 triggered the release of migration inhibitory factor (MIF). This VE-cadherin-mediated release of MIF further confirmed by ELISA with both VE-cadherin blocking antibody and siRNA technique was due to enhanced expression of MIF mRNA, which was mediated by Src kinase activation. In addition, in vivo lung vascular leak induced by VE-cadherin function-blocking antibody was partly alleviated by neutralizing MIF., Conclusions: VE-cadherin regulates MIF synthesis and release via Src kinase. Our data provide additional evidence to the concept that VE-cadherin transfers intracellular signals to coordinate the state of cell-cell adhesion with gene expression.

Rabbit monoclonal antibody (RabMAb) demonstrates higher sensitivity without sacrificing specificity than mouse monoclonal antibody (MMAb). MMAb against E-cadherin stain is heavily utilized in distinguishing ductal carcinoma in situ (DCIS) from lobular carcinoma in situ (LCIS). We aimed to compare the E-cadherin stain using RabMAb vs MMAb in distinguishing DCIS from LCIS. One hundred and seventeen in situ breast carcinomas (55 DCIS, 58 LCIS, and 4 DCIS and LCIS) were studied. Sections from a representative block of each were stained with RabMAb [EP700Y] and MMAb [36B5]. Scanned images of stained slides were compared in tandem. All DCIS cases (59/59) showed comparable staining by RabMAb and MMAb. Comparable staining was also observed in all but one case of LCIS (61/62; 98%). One case of pleomorphic LCIS showed mostly complete, weak to moderately intense membranous staining with RabMAb and fragmented, weak membranous staining with MMAb. Consistently better staining quality was observed in slides stained by RabMAb vs MMAb. RabMAb and MMAb against E-cadherin were diagnostically equivalent with the exception of one case where RabMAb may have led to diagnostic misinterpretation. However, the not insignificant cost savings and easier interpretation using RabMAb may justify the risk of misinterpretation of increased staining in rare cases, largely avertable with proper training., (© 2019 Wiley Periodicals, Inc.)

The analysis of circulating tumor cells (CTCs) provides a means to collect information about the evolving properties of a tumor during cancer progression and treatment. For patients with metastatic prostate cancer, noninvasive serial measurements of bloodborne cells may provide a means to tailor therapeutic decisions based on an individual patient's response. Here, we used a high-sensitivity profiling approach to monitor CTCs in patients with metastatic castrate-resistant prostate cancer (mCRPC) undergoing treatment with abiraterone and enzalutamide, two drugs used to treat advanced prostate cancer. The capture and profiling approach uses antibody-functionalized magnetic nanoparticles to sort cells according to protein expression levels. CTCs are tagged with magnetic nanoparticles conjugated to an antibody specific for the epithelial cell adhesion molecule (EpCAM) and sorted into four zones of a microfluidic device based on EpCAM expression levels. Our approach was compared to the FDA-cleared CellSearch method, and we demonstrate significantly higher capture efficiency of low-EpCAM cells compared to the commercial method. The nanoparticle-based approach detected CTCs from 86% of patients at baseline, compared to CellSearch which only detected CTCs from 60% of patients. Patients were stratified as prostate specific antigen (PSA) progressive versus responsive based on clinically acceptable definitions, and it was observed that patients with a limited response to therapy had elevated levels of androgen receptor variant 7 (ARV7) and the mesenchymal marker, N-cadherin, expressed on their CTCs. In addition, these CTCs exhibited lower EpCAM expression. The results highlight features of CTCs associated with disease progression on abiraterone or enzalutamide, including mesenchymal phenotypes and increased expression levels of ARV7. The use of a high-sensitivity method to capture and profile CTCs provides more informative data concerning the phenotypic properties of these cells as patients undergo treatment relative to an FDA-cleared method.

Targeting microenvironmental factors that foster migratory cell phenotypes is a promising strategy for halting tumor migration. However, lack of mechanistic understanding of the emergence of migratory phenotypes impedes pharmaceutical drug development. Using our three-dimensional microtumor model with tight control over tumor size, we recapitulated the tumor size-induced hypoxic microenvironment and emergence of migratory phenotypes in microtumors from epithelial breast cells and patient-derived primary metastatic breast cancer cells, mesothelioma cells, and lung cancer xenograft cells. The microtumor models from various patient-derived tumor cells and patient-derived xenograft cells revealed upregulation of tumor-secreted factors, including matrix metalloproteinase-9 (MMP9), fibronectin (FN), and soluble E-cadherin, consistent with clinically reported elevated levels of FN and MMP9 in patient breast tumors compared with healthy mammary glands. Secreted factors in the conditioned media of large microtumors induced a migratory phenotype in nonhypoxic, nonmigratory small microtumors. Subsequent mathematical analyses identified a two-stage microtumor progression and migration mechanism whereby hypoxia induces a migratory phenotype in the initialization stage, which then becomes self-sustained through a positive feedback loop established among the tumor-secreted factors. Computational and experimental studies showed that inhibition of tumor-secreted factors effectively halts microtumor migration despite tumor-to-tumor variation in migration kinetics, while inhibition of hypoxia is effective only within a time window and is compromised by tumor-to-tumor variation, supporting our notion that hypoxia initiates migratory phenotypes but does not sustain it. In summary, we show that targeting temporal dynamics of evolving microenvironments, especially tumor-secreted factors during tumor progression, can halt tumor migration. SIGNIFICANCE: This study uses state-of-the-art three-dimensional microtumor models and computational approaches to highlight the temporal dynamics of tumor-secreted microenvironmental factors in inducing tumor migration., (©2019 American Association for Cancer Research.)

Tumor metastasis accounts for most tumor-associated mortality and is closely related with stromal fibroblasts in the tumor microenvironment. It was reported that fibroblasts promoted tumor metastasis through directly leading tumor cell invasion; however, inflammatory microenvironment in the growing tumor may influence the outcome. Here, we found that the cytokine IFNγ, a key immune mediator secreted by T cells, could alter mouse lung tumor associated fibroblast-leading LLC tumor cell invasion in Matrigel. The motility of fibroblasts and adhesion with tumor cells were dramatically impaired upon IFNγ stimulation. We further found that IFNγ reduced the expression of N-cadherin on the surface of fibroblasts through upregulating SMAD7 and suppressing the downstream SMAD2 phosphorylation. N-cadherin was essential for fibroblast motility and adhesions with tumor cells. Moreover, fibroblasts could promote tumor progression and the deficiency of IFNγR signaling in fibroblasts reduced liver metastasis of LLC tumor in vivo. Collectively, our results demonstrate that IFNγ inhibits fibroblast-leading tumor cell invasion by inhibiting the motility of fibroblasts and their adhesion with tumor cells. The findings indicate that inflammatory cytokines in the tumor microenvironment may regulate the fibroblast-associated tumor metastasis., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: Endothelial activation and damage is commonly observed in patients with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) and is related to development of atherosclerosis and cardiovascular diseases. Different components of the immune system seem to participate in the endothelial injury, such as generation of autoantibodies and formation of immune complexes (ICs). Microparticles (MPs) and their immune complexes (MPs-ICs) are increased in the circulation of patients with SLE and RA; therefore, we propose these extracellular vesicles could interact and modulate the function of endothelial cells. Hence, the effect of MPs and MPs-ICs from patients with SLE and RA in endothelial cells was evaluated., Methods: Macrovascular and microvascular endothelial cells were exposed to MPs and MPs-ICs from healthy donors and patients with SLE and RA. Vesicles uptake/binding, expression of adhesion molecules, cytokine and chemokine production, monocyte adherence, and alterations of endothelial monolayer were evaluated by flow cytometry and fluorescence microscopy., Results: Endothelial cells internalized MPs and MPs-ICs and increased CD54 and CD102 expression and CCL2, CCL5, and IL-6 production after the treatment with these extracellular vesicles, which led to an increase in the adherence of classic monocytes. These vesicles also induced low expression of VE-cadherin in membrane, depolymerization of actin filaments, and formation of intercellular spaces, which led to endothelial death and increased permeability after MPs and MPs-ICs exposure., Conclusions: MPs and MPs-ICs from patients with SLE and RA increase adhesion molecules expression, chemokine production, and structural alterations in macrovascular and microvascular endothelial cells. Therefore, high counts of these vesicles in patients would promote endothelial alterations and secondary tissue leukocyte infiltration.

Acute lung injury and its more severe form, acute respiratory distress syndrome, are life-threatening respiratory disorders. Overwhelming pulmonary inflammation and endothelium disruption are commonly observed. Endothelial cells (ECs) are well recognized as key regulators in leukocyte adhesion and migration in response to bacterial infection. Prolyl hydroxylase domain (PHD)-2 protein, a major PHD in ECs, plays a critical role in intracellular oxygen homeostasis, angiogenesis, and pulmonary hypertension. However, its role in endothelial inflammatory response is unclear. We investigated the role of PHD2 in ECs during endotoxin-induced lung inflammatory responses with EC-specific PHD2 inducible knockout mice. On lipopolysaccharide challenge, PHD2 depletion in ECs attenuates lipopolysaccharide-induced increases of lung vascular permeability, edema, and inflammatory cell infiltration. Moreover, EC-specific PHD2 inducible knockout mice exhibit improved adherens junction integrity and endothelial barrier function. Mechanistically, PHD2 knockdown induces vascular endothelial cadherin in mouse lung microvascular primary endothelial cells. Moreover, PHD2 knockdown can increase hypoxia-inducible factor/vascular endothelial protein tyrosine phosphatase signaling and reactive oxygen species-dependent p38 activation, leading to the induction of vascular endothelial cadherin. Data indicate that PHD2 depletion prevents the formation of leaky vessels and edema by regulating endothelial barrier function. It provides direct in vivo evidence to suggest that PHD2 plays a pivotal role in vascular inflammation. The inhibition of endothelial PHD2 activity may be a new therapeutic strategy for acute inflammatory diseases., (Copyright © 2019 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Oral squamous cell carcinoma (OSCC) is the most common malignant cancer, with high mortality rates in advanced stages. Recent studies have shown that the expression of ALPK1 mRNA and its inhibitory differentiation function are associated with cancer progression. However, the expression and clinicopathologic features of ALPK1 in OSCC remain unexplored. Herein, the authors investigated the expression patterns of ALPK1 in 39 matched OSCC patients and examined the relationship between ALPK1 protein expression and clinicopathologic factors using immunohistochemical scores. Using Western blot analysis, ALPK1 expression was found to be significantly higher in tumor tissues than that in nontumor tissues. Through an immunoreactive scoring system, a significantly higher number of advanced-stage tumor size T4 and lymph node metastasis N2 exhibited higher ALPK1 expression levels than that exhibited by T1/T2/T3 tumors and N0/N1. In addition, ALPK1 protein expression was aberrant in malignant oral cancer cell lines compared with that in pre-malignant oral epithelial cells, whereas minimal expression was observed in normal oral epithelial cells. Knockdown of ALPK1 resulted in a significant reduction in cell growth, migration, and invasion capacity in vitro. Consequently, expression of N-cadherin and vimentin decreased in ALPK1-deficient cells. Thus, these results suggest that ALPK1 serves as a potential biomarker and target for OSCC development in late stages., (Copyright © 2018 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Epithelial-mesenchymal transition (EMT) is an important mechanism in tumor progression. Snail is a transcription factor, expressed in cells which have undergone almost complete EMT and have left the tumor, and Twist is considered important in the process of metastasis, both playing a major role in EMT by indirect inhibition of E-cadherin. The study analyzed the immunoexpression of E-cadherin, Snail and Twist in 46 cases of colonic carcinomas in comparison with some histopathological prognostic factors. The quantification of reactions was done by using a composite score (CS) resulted from multiplying the percentage of marked cells with the intensity of immunostaining. The majority of cases were moderately differentiated tumors, corresponded to stage III, with vascular and perineural invasion. All cases presented positive cytoplasmic and nuclear signals for Snail and Twist. The immunostaining for both markers was intense, with the highest values of CS in G2 and G3 advanced, invasive vascular colonic carcinomas, in comparison with G1, early stage lesions. We found positive significant linear correlation of Snail and Twist expression. The results obtained indicate the implication of Snail and Twist in colonic carcinoma aggressiveness, useful aspect in the oncological evaluation of patients and guided therapy.

Activated CD8 + lymphocytes infiltrate the brain in response to many viral infections; where some remain stationed long term as memory T cells. Brain-resident memory T cells (bT RM ) are positioned to impart immediate defense against recurrent or reactivated infection. The cytokine and chemokine milieu present within a tissue is critical for T RM generation and retention; and reciprocal interactions exist between brain-resident glia and bT RM . High concentrations of TGF-β are found within brain and this cytokine has been shown to induce CD103 (integrin αeβ7) expression. The majority of T cells persisting within brain express CD103, which aids in retention through interaction with E-cadherin. Likewise, cytokines produced by T cells also modulate microglia. The anti-inflammatory cytokine IL-4 has been shown to preferentially polarize microglial cells toward an M2 phenotype, with a corresponding increase in E-cadherin expression. These findings demonstrate that the brain microenvironment, both during and following inflammation, prominently contributes to the role of CD103 in T cell persistence. Further evidence shows that microglia, and astrocytes, upregulate programmed death (PD) ligand 1 during neuroinflammation, likely to limit neuropathology, and the PD-1: PD-L1 pathway also aids in bT RM generation and retention. Upon reactivation of quiescent neurotropic viruses, bT RM may respond to small amounts of de novo-produced viral antigen by rapidly releasing IFN-γ, resulting in interferon-stimulated gene expression in surrounding glia, thereby amplifying activation of a small number of adaptive immune cells into an organ-wide innate antiviral response. While advantageous from an antiviral perspective; over time, recall response-driven, organ-wide innate immune activation likely has cumulative neurotoxic and neurocognitive consequences.

Kyasanur Forest disease virus (KFDV) is a highly pathogenic tick-borne flavivirus enzootic to India. In humans, KFDV causes a severe febrile disease. In some infected individuals, hemorrhagic manifestations, such as bleeding from the nose and gums and gastrointestinal bleeding with hematemesis and/or blood in the stool, have been reported. However, the mechanisms underlying these hemorrhagic complications remain unknown, and there is no information about the specific target cells for KFDV. We investigated the interaction of KFDV with vascular endothelial cells (ECs) and monocyte-derived dendritic cells (moDCs), which are key targets for several other hemorrhagic viruses. Here, we report that ECs are permissive to KFDV infection, which leads to their activation, as demonstrated by the upregulation of E-selectin, intercellular adhesion molecule 1, and vascular cell adhesion molecule 1 at the mRNA and protein levels. Increased expression of these adhesive molecules correlated with increased leukocyte adhesion. Infected ECs upregulated the expression of interleukin (IL)-6 but not IL-8. Additionally, moDCs were permissive to KFDV infection, leading to increased release of IL-6 and tumor necrosis factor-α. Supernatants from KFDV-infected moDCs caused EC activation, as measured by leukocyte adhesion. The results indicate that ECs and moDCs can be targets for KFDV and that both direct and indirect mechanisms can contribute to EC activation.

Plasmacytoid dendritic cells (pDCs) play a key role in antiviral responses by producing type-1 IFNs. However, recent studies showed that pDCs induce immune suppression and promote tumor growth in human ovarian cancer and myeloma. The molecular mechanisms underlying pDC acquisition of these properties are unknown. Here we show that human pDCs activated by CpG inhibited growth and induced apoptosis in myeloma cells via secreted IFN-α, but direct contact with myeloma cells converted pDCs into tumor-promoting cells by suppressing pDC IFN-α production. E-cadherin, expressed on both myeloma cells and pDCs, mediated these effects via a homophilic interaction - activation of E-cadherin signaling upregulated and activated TNFAIP3 to interact with TLR9, resulting in TLR9 ubiquitination and degradation, and inhibition of IFN-α production in pDCs. These findings were supported by an in vivo study in which pDC depletion induced tumor regression and better survival in the Vk*MYC myeloma mouse model. Furthermore, IFNAR1 expression level positively correlated to overall survival of patients with multiple myeloma (MM), and the IFN-α level in patient bone marrow was significantly lower than that in marrow of healthy individuals. This study reveals a novel mechanism underlying how MM tumors educate pDCs in their microenvironment and provides new targets for improving the treatment of MM.

Cadherin-2 plays a fundamental role during zebrafish CNS and heart morphogenesis. Profiling zCdh2 expression via antibody staining is essential to achieving better understanding of its role during zebrafish development. Generation of recombinant human antibodies to zCdh2 by phage display was used to identify monoclonal antibodies with reduced unspecific binding patterns when compared to available commercial antibodies. Specificity was profiled using flow cytometry of wild type, zCdh2-defective mutant and zCdh2-GFP zebrafish cells. The epitopes recognized by the novel antibodies were mapped to peptides located in the first or second extracellular domains of zCdh2. These antibodies allowed improved observations of the spatial distribution of zCdh2 from imaging of whole mount zebrafish preparations. Since the generated antibodies are sequence defined, they can always be reconstituted from the information stored in the respective computer file, securing future reproducibility of respective experiments. The results further suggest that sequence defined antibodies with specificities thoroughly controlled by flow cytometry and genetic antigen-defective mutants or knockouts can substantially reduce the risk of misleading, false-positive results in whole mount imaging and other assays, and thus can improve the scientific value of such assays., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Sertoli cells were isolated from newborn calves and cultured in a medium supplemented with 0, 0.25, 0.50, 0.75, and 1.00 mg/L of sodium selenite to study their immune stimulatory effect, influence on cell's viability, and expression of blood-testis barrier proteins (occludin, connexin-43, zonula occluden, E-cadherin) using quantitative PCR and western blot analyses. Results showed that medium supplemented with 0.50 mg/L of selenium significantly (P < 0.05) promoted cell viability, upregulated toll-like receptor gene (TLR4), anti-inflammatory cytokines (IL-4, IL-10, TGFβ1), and expressions of blood-testis barrier proteins, and modulated expressions of pro-inflammatory cytokines (TNF-α, IL-1β, IFN-γ). Sertoli cells grown in culture medium supplemented with 0.25 mg/L of selenium significantly upregulated TLR4, IL-4, IL-10, TGFβ1, and blood-testis barrier proteins compared to the control group. Sodium selenite supplementation at 0.75 and 1.00 mg/L levels was cytotoxic and temporarily downregulated the expression of blood-testis barrier protein within 24 h after culture; however, commencing from 72 h post culture, increased cell viability and upregulation of expression of blood-testis barrier proteins were observed. In conclusion, the results of this study showed that selenium supplementation in the culture medium up to 0.50 mg/L concentration upregulates immune genes and blood-testis barrier constituent proteins of bovine Sertoli cells.

The inhibitor for the homophilic dimerization of P-cadherin was discovered by SPR-based screening using fragment compounds. Our SPR assays identified a specific P-cadherin binder, which was able to inhibit the cell adhesion of living CHO cells that expressed P-cadherin.

Adoptive transfer of T cells engineered with a cancer-specific T cell receptor (TCR) has demonstrated clinical benefit. However, the risk for off-target toxicity of TCRs remains a concern. Here, we examined the cross-reactive profile of T cell clone (7B5) with a high functional sensitivity for the hematopoietic-restricted minor histocompatibility antigen HA-2 in the context of HLA-A*02:01. HA-2 pos Epstein-Barr virus-transformed B lymphoblastic cell lines (EBV-LCLs) and primary acute myeloid leukemia samples, but not hematopoietic HA-2 neg samples, are effectively recognized. However, we found unexpected off-target recognition of human fibroblasts and keratinocytes not expressing the HA-2 antigen. To uncover the origin of this off-target recognition, we performed an alanine scanning approach, identifying six out of nine positions to be important for peptide recognition. This indicates a low risk for broad cross-reactivity. However, using a combinatorial peptide library scanning approach, we identified a CDH13-derived peptide activating the 7B5 T cell clone. This was confirmed by recognition of CDH13-transduced EBV-LCLs and cell subsets endogenously expressing CDH13, such as proximal tubular epithelial cells. As such, we recommend the use of a combinatorial peptide library scan followed by screening against additional cell subsets to validate TCR specificity and detect off-target toxicity due to cross-reactivity directed against unrelated peptides before selecting candidate TCRs for clinical testing., (Copyright © 2018 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

We propose a new cadherin family classification comprising epithelial cadherins (cadherin 17 [CDH17], cadherin 16, VE-cadherin, cadherin 6 and cadherin 20) containing RGD motifs within their sequences. Expression of some RGD cadherins is associated with aggressive forms of cancer during the late stages of metastasis, and CDH17 and VE-cadherin have emerged as critical actors in cancer metastasis. After binding to α2β1 integrin, these cadherins promote integrin β1 activation, and thereby cell adhesion, invasion and proliferation, in liver and lung metastasis. Activation of α2β1 integrin provokes an affinity increase for type IV collagen, a major component of the basement membrane and a critical partner for cell anchoring in liver and other metastatic organs. Activation of α2β1 integrin by RGD motifs breaks an old paradigm of integrin classification and supports an important role of this integrin in cancer metastasis. Recently, synthetic peptides containing the RGD motif of CDH17 elicited highly specific and selective antibodies that block the ability of CDH17 RGD to activate α2β1 integrin. These monoclonal antibodies inhibit metastatic colonization in orthotopic mouse models of liver and lung metastasis for colorectal cancer and melanoma, respectively. Hopefully, blocking the cadherin RGD ligand capacity will give us control over the integrin activity in solid tumors metastasis, paving the way for development of new agents of cancer treatment., (Copyright © 2018 Elsevier B.V. All rights reserved.)

It is theorized that toxic agents are transported from the hyperpermeable gut of burn victims through the lymph, to the systemic circulation, causing global injury. We believe that immune cells respond to leakage of "toxic lymph" following trauma causing the attraction of these cells to the perilymphatic space. To test this, we utilized a model of burn on rats to examine changes in a single immune cell population associated with mesenteric lymphatic dysfunction. We examined the ability of serum from these animals to increase permeability in lymphatic endothelial monolayers and disrupt cellular junctions. We also treated burn animals with doxycycline, an inhibitor of microvascular permeability, and observed the effects on immune cell populations, morphometry, and lymphatic endothelial permeability. Burn injury increased the number of MHCII + immune cells along the vessel (>50%). The size and shape of these cells also changed significantly following burn injury. Serum from burn animals increased lymphatic endothelial permeability (∼1.5-fold) and induced breaks in VE-cadherin staining. Doxycycline treatment blocked the accumulation of immune cells along the vessel, whereas serum from doxycycline-treated animals failed to increase lymphatic endothelial permeability. The size of cells along the vessel in doxycycline-treated burn animals was not affected, suggesting that the cells already present on the lymphatic vessels still respond to substances in the lymph. These findings suggest that factors produced during burn can induce lymphatic endothelial barrier disruption and lymph produced during traumatic injury can influence the attraction and morphology of immune cell populations along the vessel.

Strong evidence exists supporting the important role T cells play in the immune response against tumors. Still, the ability to initiate tumor-specific immune responses remains a challenge. Recent clinical trials suggest that bispecific antibody-mediated retargeted T cells are a promising therapeutic approach to eliminate hematopoietic tumors. However, this approach has not been validated in solid tumors. PF-06671008 is a dual-affinity retargeting (DART ® )-bispecific protein engineered with enhanced pharmacokinetic properties to extend in vivo half-life, and designed to engage and activate endogenous polyclonal T cell populations via the CD3 complex in the presence of solid tumors expressing P-cadherin. This bispecific molecule elicited potent P-cadherin expression-dependent cytotoxic T cell activity across a range of tumor indications in vitro, and in vivo in tumor-bearing mice. Regression of established tumors in vivo was observed in both cell line and patient-derived xenograft models engrafted with circulating human T lymphocytes. Measurement of in vivo pharmacodynamic markers demonstrates PF-06671008-mediated T cell activation, infiltration and killing as the mechanism of tumor inhibition.